Proc. Nati. Acad. Sci. USA -j_1.! ,^; tarn [Clint em -; S Vol. 83, pp. 2619-2623, April 1986 Immunology

Cloning and sequence analysis of complementary DNA encoding an aberrantly rearranged human T-cell y chain (cDNA/DNA sequence) DENO P. DIALYNAS*, CORNELIS MuRREt, THOMAS QUERTERMOUSt, JEREMY M. Boss*, JEFFREY M. LEIDEN*, J. G. SEIDMANt, AND JACK L. STROMINGER* + *Department of Biochemistry and , Harvard University, 7 Divinity Avenue, Cambridge, MA 02138; and tDepartment of , Harvard Medical School, 45 Shattuck Street, Boston, MA 02115 Contributed by Jack L. Strominger, December 16, 1985

ABSTRACT Complementary DNA (cDNA) encoding a citrate) at 42°C for 16 hr, using a nick-tinslated Pst I/BamHI human T-cell y chain has been cloned and sequenced. At the fragment encoding part of the humanrT-celliy-chain CI exon junction of the variable and joining regions, there is an (4) as a probe. The filters were then wd hed twice at 'room apparent deletion of two in the human cDNA temperature with 2x SSC, 'followed' byt washes at'53TC sequence relative to the murine y-chain cDNA'sequence, with 0.2x SSC. resulting simultaneously in the generation of an in-frame stop Construction of the HPB-MLT cDNA LiIrary. Total RNA codon and in a translational frameshift. For this reason, the was isolated from logarithmic-phase H1iBHMLT cells by'the sequence presented here encodes an aberrantly rearranged lithium chloride/urea method' (5).' y(34 RNA was pre- human T-cell y chain. There are several surprising differences pared by one cycle of oligodTl)-cedlliose ity chromatog-' between the deduced human and murine Y-chain amino acid' raphy. First-strand cDNA was 'synthesi'zed with avian sequences. These include poor homology in the variable region, myeloblastosis reverse'trans ta'se, using as template poor homology in a discrete segment of the constant region 30',ug of HPB-MLT poly{A)+VRNUiat'had first'been precisely bounded by the expected junctions of exon- CII, and denatured by heating at 70°C for 3 win. Serond-strand cDNA the presence in the human sequence of five potential sites for synthesis using the Klenow fragmeptef.;59A polymerase'I N-linked glycosylation. followed denaturation of the first-sgra'nd reaction product with 12.5 mM CH3HgOH. The cDNA hai'pit loop was then The murine T-cell y-chain unit is constructed cleaved with nuclease Si,'After which te"cDNA was methyl- through T-cell-specific somatic juxtaposition of germ-line ated with EcoRI methylase, blunt-enaeK usi'ng the' Klenow' variable (V), joining (1), and constant (C) region elements (1). ftagment ofDNA polym,'erase I, and ligatedWto'EcoRI linkers. Both in this context and at the level of amino acid sequence The cDNA was then digested with ko'IA hromatographed homology, the y chain is similar-to the a and f3 chains of the on Sepharose CL-4B both' to ren~ove free, linkers and to antigen receptor on mature T cells, more generally referred size-fractionate the cDNA, and finally liged to EcoRI-cut to as the T-cell receptor. The -y-chain locus is distinguished XgtlO vector (6). The product,'of theqlgation reaction 'was in part by the apparent paucity of V- 'and J-region elements; packaged, giving 3.3 5 106 independent recombinant phage, in fact, the greatest apparent variability resides at the V-J as determined on Eschirichia coli' 600',with an average junction, generated by unknown mechanisms (2). insert size of 1100 base pairs. This' libr'ary designated' The function of the y chain, which has yet to be identified MLT:XgtlOA, was carried through one cycle of amplification at the level, is unknown. To determine which prop- on E. coli C600Hfl1 such that 85% of the resultant phage erties of the y chain are essential to its function, and thereby contain inserts. gain insight into that function, we have initiated a character- Screening the HPB-MLT cDNA'Libraoryf6r the Human ization of the human y-chain locus. We report here the T-Cell yChain. An aliquot of cDNA ILT:Xgt1OA was isolation and sequence analysis of complementary DNA plated on E.. coli C600If1+ at a, density,''of 3.5 x 104 (cDNA) clones encoding an aberrantly rearranged- human plaque-formingunits per 150-mm plate. Itiujlicate fgters were prepared from 10 plates' and 'screened. T ~y1ridization -was, T-cell y chain. carried out in 50% formamnide/6x SSC at 4t°Cfor 16 hr, using a nick-translated Pst I/BamHI fragmentericoding part of the MATERIALS AND METHODS human T-cell -chain CI exon (4) aF probe.The filters were then washed twice at room temperature With 2x SSC, Enzymes. Restriction enzymes were from New England followed by two washes at 530C wid 2'0!-x SSC. Positive, Biolabs, Bethesda Research Laboratories, and Boehringer recombinant'phage were plaque-purified.'I-Tw6 clones, Ty1' Mannheim. Avian myeloblastosis virus and T-2, have been charaiterized'e'tensively. was from Life Sciences. T4 DNA ligase, EcoRI methylase, DNA Sequence Analysis The inserts'sr h Ty-1 4nd Ty2 and EcoRI linkers were from New England Biolabs. The were subcloned into the EcoRI site bfflUCO(7) to give pTl- Klenow fragment of DNA polymerase I was from Bethesda and pTy-2, respectively. The insert in pToy4 was sequenced Research Laboratories. Nuclease S1 was from Boehringer by the chemical-degradation method t(8)Q+ragments to be Mannheim. sequenced were labeled---at . the 3' end-ising the Klenow RNA Blot Analysis. Poly(A)+ RNA was denatured by fragment of DNA polymerase.I (3),. Tq-sq~once from the Pst formaldehyde, electrophoresed, and transferred onto a ni- I and Acc I sites, EcoRL/Pst I anj >E Rj/cc I fragments trocellulose filter (3). Hybridization was carried out in 50o from pTy-1 were subcloned, into.pJC13,(7) and then se-, formamide/6x SSC (lx SSC = 0.15 M NaCl/0.015 M Na quenced from the HindII site in thq pVIj`3,polylinker. The. internal BamHI fragment ofpTy-1 wa ,4,lAped into pUC13 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: V, J, and C vi, bqjpij'p g, and constant, in accordance with 18 U.S.C. §1734 solely to indicate this fact. regions.

2619 Downloaded by guest on October 1, 2021 2620 Immunology: Dialynas et al. Proc. Natl. Acad. Sci. USA 83 (1986)

_ X

...... ,,....'...-/~uS/,/ -28S R K Bx B HB P N Ac 0oo RK Bx BH BEP N Ac R

- 18S FIG. 1. RNA blot analysis of formaldehyde-denatured poly- * (A)' RNA from B-cell line LAZ FIG. 2. Both the restriction map and sequencing strategy are I 509 and T-cell line HPB-MLT given here for the inserts in pTt-1 and pT-t-2. Restriction sites for expression of the human T- indicated are EcoRI (R), Kpn I (K), BstXI (Bx), BamHI (B), HincHI cell -tychain transcript. (H), Ava 11(A), Pst I (P), Nco I (N), andAcc I (Ac). The approximate junctions of the leader peptide *, V cm, J m, C , and 3'- untranslated o regions are indicated. The insert in pTv-1 was and then sequenced both from the EcoRI and from the sequenced by the chemical-degradation method (8) (e). The insert in HindIII sites in the pUC13 polylinker. To sequence from the pTy-2 was sequenced by the dideoxy chain-termination method (9, Kpn I site directly in pT-l, fragments were generated by 10) (o). bp, Base pairs. using Asp718 (Boehringer Mannheim). The insert in pTy2 was sequenced by the dideoxy chain-termination method (9, 10). Restriction fragments as indicated were subcloned into T-cell 'y chain, it was essential to identify a human cell line either M13 mp8, M13 mp9, M13 mpl8, or M13 mpl9 (7). M13 expressing the y-chain transcript. To this end, a panel of mp8 and M13 mp9 were from Amersham, whereas M13 mpl8 human T-cell lines was screened by RNA blot analysis, using and M13 mpl9 were from New England Biolabs. Sequencing a Pst I/BamHI fragment encoding part ofthe human -t-chain was carried out using dATP [a-35S] and a sequencing kit from CI exon (4) as probe. By this criterion, the T-cell line Amersham. HPB-MLT was found to express the y-chain transcript or a transcript highly homologous to it, whereas the B-cell line RESULTS LAZ 509 did not (Fig. 1). Other T-cell lines analyzed by RNA RNA Blot Analysis of Human T-CeUl Lines for Expression of blot gave either no signal at all or an ambiguous signal (data the -Chain Transcript. To clone cDNA encoding the human not shown). The apparent size of the hybridizing RNA is 1.6 I-20t leader peptide -lI +1 9 CCC ATC CCGC TGC CCC CTA CTC CTG CTT CTA CCT TTC CTG TCT CCT CCC ACT CAC MA TCT TCC MC TTC GM CCC ACA ACC MG TCA CTC MLT H R W A L L V L L A F L S P A S Q K S S N L E C R T K S V * * a * * * 90 H L L L R W P T S C CL W V P C L C Q L E Q T L S V MC CTT CCC ATC CTC CTC CTG ACA TCC TTC ACC TCC TGC TCC CTC TGC CIT ITT GGCCTTCCGCAG CTG CAG CM ACT GM TrA TCG CTC 2C (-L XI< V 37 ACC ACC CAC AC? CGG TCA TCT CCT GM ATC ACT TGC CAT CTT ACT CTA ACA MT ACC TTC TAC ATC CAC TCC TAC CTA CAC CAG MLT TR Q T C S S A E I T caD L TV T N T F Y I 1 W YL H Q a * * * * * * ** * 174 T R E T D E N V Q I S 13I V Y L PY P S N T A I H W Y R Q ACC ACA GAG ACA CA? GAG AAT GTC CM ATA TCC TCT ATA CTT TAT CTT CCA TAT TTC TCC MC ACA GCT ATA CAT TGC TAC CCC CM v 67 GAG CCC AAG GCC CCA CAC CCT CTT CTC TAC TAT CAC CTC TCC ACT CCA ACC CAT CTC TTG CM TCA GCA CTC ACT CCA CCA MG TAT TAT MLT E CK A P Q RL L Y Y D V. S T A R D VL E S C L S P c K Y Y * * * 264 K T N Q Q F E Y L I Y V A TN Y N Q R F L C C K H K K I E A AM ACA MT CM CAG TTT GAG TAT CTA ATA TAT CTC CdA ACA MC TAC MT CAA CGA CCC TTA CCA GCC MC CAC MA AM ATT CM CCA 2C _.. v 97 ACT CAT ACA CCC AGC ACC TCC ACC TCG ATA TTC AGA CTC CAA AAT CTA ATT CM AT CAT TCT COC CTC TAT TAC TCT CCC ACC TCG CAC MLT T 11 T P R R W S W I L R L Q N L I IN DS C V Y Y A T W D * * * * * * * 354 S K D F K S S T S T L E I N Y L K K E D E A. T Y Y M A V W H AGT MA GAT TTT AM ACT TCT ACC TCA ACC TTG CM ATA AAT TAC TTG MC AA CM CAT CM CCC ACC TAC TAC TCT GCA CTC TCC ATG 2C v 125 ACC C MA MT TAT TAT MC MA CTC TIT CCC ACT GCA ACA ACA CTT CTT CTC ACA CAT AA CAA CTT CAT CCA CAT CTT TCC MLT R KN Y Y K K L PC S C TT L V V T D K QL D A D V S * * * * * * * * * * ** * * * 436 RY S S C F H K V F A E c T K L I V I P S D K R L D A D I S ACA TAT ACC TCC CCC TTT CAC MC CTA TTT CCA CM CCA ACA MG CTC ATA CTA ATT CCC TCCGAC AA AGG CTT CAT CCA CAC ATT TCC 2C 4k-?,pIk J >1 + CI 155 CCC MC CCC ACT ATT TTT CTT CCT TCA ATT CCT CAA ACA AM CTC CAG MG CCT CA ACA TAC CTT TCT CTT CTT GAG mA TT TTC CCA MLT P K P T I F L P S 'I A E T K L Q K A G T Y L j L L E K F F P * * ** ** *** * * * * * * * * * * * * * * * * * 526 P K P T I F L ? S V A E T N L H K T C T Y L I L L E K FF P CCC MG CCC ACT ATT TTT CTT CCT TCT CTT CCT CM ACA MT CTC CAT AAC ACT CCC ACA TAC CTT TCT CTC CTT CM MGC TC TTT CCC 2C

FIG. 3. (Figure continues on the opposite page.) Downloaded by guest on October 1, 2021 Immunology: Dialynas et al. Proc. Natl. Acad. Sci. USA 83 (1986) 2621

185 CAT ATT ATT MG ATA CAT TCC CM GMMM MC ACC MC ACG ATT CTG CGA TCC CAG GAG CCC MC ACC ATC MC ACT MC CAC ACA TAC MLT D I I K I H1 W Q EK K S N T I L C S Q E c NT N K T IN D T Y * * * * * * * * * * * ** * * * * * 616 D V I R VY W K E K N C N T I L D S Q E C D T L K T K G T Y CAT CTC ATA ACC CTG TAT TCG MA GM AAG MT CCC MT ACT ATC CTG GAC TCC CAG GM CCC CAT ACG CTC MC ACT MG CCC ACA TAC 2C CI 215 ATC AM TTT ACC TCG TTA ACC GTG CCA CM GAG TCA CTG GAAAC CM CAG AGA TCT ATC CTC ACA CAT GAG MT MT AM MGCCA ATT MLT M K F S W L T V F E E S LD K E H R ff I V R H E N N K N G I * ** * ** * ** * * * * * * * * * * * * * 706 M KF S W L T V P F R A H C K E H S. f I V K H E N N K C C A ATC AAC TTT ACC TCC CTT ACT CTG CCT CM ACCCGA ATC CCC MA GAG CAC ACT TCT ATT CTC AM CAT GAG MG MC MA CCA CCA CCA 2C CI 245 CAT CM CM ATT ATC TT CCT CCA ATA MC ACA CAT GTC ACC ACA CTG CAT CCC MA CAC ACT TAT TCA MA CAT GCA MT CAT CTC ATC lILT D Q E I I F P P I K T D V T T VD P K D S Y S K D A N D V I * * * * * * * * * * 796 D Q E 1FF P S I K K V A T T (1 W Q D K N CAT CM GAG ATT TTC TTC CCT TCA ATA MG AAA CTT CCT ACA ACT TCC TCG CM CAT MA MT 2C - ~~~CI :14 - CII 275 ACA ATC CAT CCC AM CAC MT TGC TCA MA CAT CCA MT CAT ACA CTA CTC CTC CAG CTC ACA MC ACC TCT CCA TAT TAC ATC TAC CTC MLT T M D P K D IN S K D A IN D T L L L Q L T IN TA YY M. YL * * * * * ** * * * * 886 D V L Q F Q F T S T S A Y Y T Y L CAT CTG CTG CAG TTC CAG TTC ACG ACC ACC TCT CCG TAC TAC ACC TAC CTC 2C CII >14 CIII traqsembrane > I cytoplasmic CTC CTC CTC CTC MC ACT CTG CTC TAT TTT CCC ATC ATC ACC TCC TCT CTG CTT CCA ACA ACG GCT TTC T TC C MT CCA GAG MA TCA MLT L LL L E S V V YF A I I T C CLL C R T A F C N C E K S * * * * * * * * * ** ** * * * * * * 976 L L L [i] S V I Y L A I I S F S L L RR T S V c N E K K S CTC CTG CGC CTC MC ACT CTC ATC TAC TTC CCC ATC ATC ACC TTC TCT CTG CTT ACA ACA ACA TCT CTC TCT CCC MT GAG MC MG TCC 2C CIII )

TM CACACCCTCCCACAACCACCCCATCTTTTCCTCATCGGTTATTCTCCCTACMCCCTCTJCTCACCATCTACTTCCCCTTTTrCTTCCnTTTCCCCAmGACTTCTGCATTC MLT 1094 TM AGAMCCACTGCTCCTACACCAAMCTCACCTCCAT1TCATCCTCACTGCCATAMAGCTGCTTMCCCGCAACCACATGCCTJCTCTTGTTGCCTTTCACTTCrATAACTCCCTC 2C

HLT 1213 ACTCATCTTCCATAAACATrTTCTCAACT-ruiC ATCCAATTrCAGCAACrTTT7TAAACTCAACTCACCTTCrTCCrCATTCCATCCACTCCAGAACTCCCCrCCCCCACAAGCCTCA 2C

TTCCACAGCrCCTCCACCCAACCCAAATACCCCCrGCrATAGTGTAGACAGCCTCCGGC7'TCrAGCCTTCTCCCTCTCrTAGTGTTCTTTAATCAGATAACTGCCTGGAAGCCTTTCAT MLT 1332 MCATTAAATTCTACTACCCATACCCTACACrTI'TACGCATCCGCCTT 2C

TJTACACCCCGCTGMGCACTCTTCTTTCCTACTTGMTTATGTGCTCTGTTTTTCCGCTMTAMCCAAMTMATTTAAAAAMTGAMA MLT 1421

FIG. 3. Comparison of the human and murine T-cell v-chain cDNA sequences. The sequence and corresponding amino acid sequence ofpTy1/2 (from the human T-cell line HPB-MLT) are compared with the nucleotide sequence and corresponding amino acid sequence ofpHDS4 (from murine cytotoxic T-cell line 2C) (11). Nucleotide position for sequence pTr1/2 is indicated to the right of the sequence; amino acid position is indicated above the sequence. Amino acids are designated by the one-letter code. In the alignment of the amino acid sequences, several gaps have been introduced as shown. Aligned amino acids are indicated by an asterisk. The approximatejunctions of the leader peptide, transmembrane, and cytoplasmic domains are indicated above the sequence, by analogy with those published for pHDS4 (11). The established (1) exon junctions for pHDS4 are indicated below the sequence. The polyadenylylation signal (AATAAA) is underlined. Cysteines believed to be involved in either intrachain or interchain disulfide linkage, potential sites for N-linked glycosylation (N-X-S or N-X-T), and the conserved lysine residue in the transmembrane region are boxed. Note that at the V-J junction there is an apparent dinucleotide deletion in the human cDNA sequence relative to the murine cDNA sequence, resulting simultaneously in the generation of an in-frame stop codon (TAA, overlined) and in a translational frameshift.

kilobases. By flow cytometric analysis, HPB-MLT has char- clones, Tv-i and Tv-2, have been characterized extensively. acteristics both of an immature (coexpression of T4 and T8) DNA Sequence Analysis of the HPB-MLT T-Cell yChain and of a mature (expression of T3) thymocyte (data not cDNA. The inserts from Ty-1 and Ty-2 were subcloned into shown).34 the EcoRI site of pUC9 to give pTy-l and pTv-2, respective- Screening the HPB-MLT cDNA Library for the Human ly. Initially, the insert in pT-1l was sequenced in entirety. T-Cell y Chain. The HPB-MLT cDNA library MLT:XgtlOA Because certain features of the pT-1 sequence were unex- was screened with the same probe used in the RNA blot pected, as discussed below, cDNA clone pTv-2 was also analysis. Representation of the y chain in this library was sequenced in entirety. The sequencing strategies for pTv-1 found to be 0.02%, 1/10th that which has been found in this and pTv-2 are shown in Fig. 2. library for the T-cell receptor chain (data not shown). The sequence of pTv-1 and pTv-2 is given in Fig. 3. The Positive recombinant phage were plaque-purified. Two pTv2 sequence begins at nucleotide position 17 in the pTv-1

741 A TG T C A|C|C A C A G T G G A T C CC AAA G A C A G A T|T C A A A A G A T G C A A A T G 788 789 A TG T C AIT[C A C AIA|T GG A T C C C A A A G A C A8A3GG6TC A A A A G A T G C A A A T C 836

FIG. 4. Putative exon CII for the v-chain cDNA from HPB-MLT consists precisely of a direct repeat of a 48-nucleotide sequence. Downloaded by guest on October 1, 2021 -2622 Wf lbgy Dildynask et al.PProc. Natl. Acad. Sci. USA 83 (1986)

K N YY:ZjKsirT GStG T T L- V;T hy: pTy-1 junction a dinucleotide deletion relative to pHDS4, with ..S CFt A- K, I iti)[ P S, my: pHDS4 similar consequences. As the origin of this pHDS4 dinucleo- -* S(3i --LL-"-K- -AT - ma: TMll tide is unknown (1), the basis for its absence- in pTy-1/2 L T - - - - -K V I -L P ma: pHOK58 remains obscure; however, it has been conjectured that this S S AS -II I .-- - - -R S IR ha: pY14 pHDS4 dinucleotide may have arisen either through terminal *,QY 7, P - --RT - L nt: pHDS11 transferase activity or through utilization of an as yet un- *Q9*Q Xtr-, -RL.- m: p2B4.71 identified diversity-region exon (1). Southern blot analysis-of -T, V Nh: YT35 the y-chain locus in HPB-MLT (12) and restriction analysis v P --R-.L L ha:4D1 of three other y-chain cDNA clones from library MLT:Xgt- 1OA (data not shown) together suggest, but do not prove, that FIG. 5. CompgrV4 amino..-l--Racid sequence of the pTy-1/2 J there exists but a single -chain transcript in HPB-MLT, region with the auiinp0,apid sequence ,of human (h) or murine (i) J regions 'R.EjTQY.--T-cell a- or /3-chain cDNAs. The represented by the pTy-1/2 sequence. express^41jn recepto-r one-letter amino acid code is used. Amino acids shared between a Even more striking is the discontinuity in amino acid {given sequencesanrd pT-lk,2 are-indicated by a dash in the given homology between pTy-1/2 and pHDS4 C regions. Hom'ol- sequence. The seqtiences presented here, except for pTy-1/2, have ogy is high at both the 5' and 3' ends, but it is poor in a discrete * bee'n.,published-elsewhere. as indicated- pHDS4 (11), TT11 (13), segment-precisely bounded by the expectedjunctions ofexon pHDS58 (16), pY14(17)X pHDS11 (11), p2B4.71 (14), YT35 (18), 4D1 CII. The putative pTy-1/2 CII exon is 3 times the length of (15).: 1i.itn" d ' the pHDS4 CII exon; moreover, close analysis reveals that it consints precisely of a direct repeat of a 48-nucleotide sequence. Aprt i the pt'-2 sequence is identical to sequence (Fig. 4). The relationship at the nucleotide' level that for pTy-, exceptthis,that cytosine at position 805 in pTy-1 between the putative pTy1/2 CII exon and the pHDS4 CII is replaced by in pTy-2. This nucleotide difference, exon is obscure. At least one consequence of this difference which most likely repredents an error by reverse transcript- is that nowhere in the putative pTy-1/2 CII exon is there a Ase during'- co'nstuctionA'of thie CDNA library, destroys a counterpart to the cysteine found in the pHDS4 CII exon. It BamHI site in pTr2 and creates an Ava II site in pTy1. The is unclear what selective force(s) operate on the y chain, such rmpre representative 'te ence is pT-2, in that six other that the divergence between humans and mice observed for y-chain cDNA'e6~iesis6taited forn libray MLT:XgtlOA also this segment of the constant region is tolerated. lack an Ava, II ity restrction analysis (data not shown). As indicated, the pT'y1/2 and pHDS4 constant regions are The sequence given n Fig. 3 is referred to below as pT-1/2. highly homologous in the' segments corresponding to exons CI (74%) and CIII'(64%) ofpHDS4. Several ofthe conserved amino acids in exon CIII are of particular interest. These DISCUSSION include the Y-X-X-L-L-X-K-X-X-X-Y sequence in the transmembrane domain and the cysteine in the cytoplasmic There are a number of points to be made about the pTy-1/2 domain. As previously pointed out (1), the conservation of 'sqequence. .NoSo l'at at the V-J jundfion there is an apparent this transmembrane sequence extends to that of the /8 chain deletion of two iicleotides in the human cDNA sequence of the T-cell receptor. The conservation of the cytoplasmic relative to the pyurkqe y-chain cDNA sequence pHDS4 (11), cysteine is especially interesting in light of the apparent lack resulting sirnultantslyfin the generation of an in-frame stop of conservation of the cysteine found in murine exon CII. It codon and in a translational frameshift. The pT-1/2 se- may be that, at least for the human y chain, any interchain quence thus encoders anaberrantly rearranged y.chain. In this disulfide linkage is'effected through the cytoplasmic cysteine. context, pT-1/2 is formally analogous to the murine y-chain We have shown elsewhere that there exist two Y-chain cDNA clone pHISM41(2), which also sustains at the V-J C-region segments in the human genome, designated C1

.. / .SH 8 ,A .. .. 'i4'QSVTQLGSHV'SVS EGALVLLRCNYSSS VPPYLFWY VQYPNQGLQrrL ,S

KY TS A.ATLVKGINGFE-AEFKKSETSFH LTK PSAHMSDAAEYFCAV ha:pY14 *;*..e! ',YDVS.TARDVLESGLSPGKYYTHTPRRWSWILRLQN LIENDSGVYYCAT hy:pTy-1 f..,. e£, *z * * * .* * * .* * * * *,* YFNNNV PIDDSGMPEDRFSAKMPNASFSTLKIQPS EPRDSAVYFCAS he:YT35

!. ',' .. Stearr'....'

k.lt 8.Q SV T Q PDARVTVStGASLQLRCKYSYSATPY LFWY Vi5YPRQGLOLL P-y''1- N LEGRT'KSVTRQTGSSAEITCDLTVTNTFY IHWYLHQEGKAPQRLL -DR * * * * ,j,,. l:DSij.DMKVTQMSRYLIKRMGENVLLECG QDMSH ETMYWY RQDPGLGL QLI

LKYYSGD PVVQGVNGFEAEFSRSNSSFHrRKArVHWSDSAVYFCAV m:pHD58 * * * ** ** ** * * YYDVSTARDVLESGLSPGKYYTHTPRRWSWILRLQNLIENDSGVYYCAT hy:P1f-1 * * * * * * * * ** ** YISYDVD SNSEGDIPKGYRVSRKKRE HFSLILDSAKTNQTSVYFCAQ mo:pnSU FIG. 6. Comparison of the amino acid sequence of the pTy-1/2 V region with the amino acid sequence of human (h) or murine (m) V regions expressed in T-cell receptor a- or 3-chain cDNAs. The one-letter amino acid code is used. The alignment adopted for pY14 and YT35 is that used previously (17), as is the alignment adopted for pHDS58 and pHDS11 (16). Amino acids aligned between pT-1/2 and either of the two companion sequences are indicated by an asterisk. Amino acids aligned between the two companion sequences are indicated by underlining or overlining. Downloaded by guest on October 1, 2021 Immunology: Dialynas et al. Proc. Natl. Acad. Sci. USA 83 (1986) 2623 and C2 (4, 12). The CI exon sequenced previously and the locus, a determination of the extent and nature of variability pT-1/2 CI exon belong to the C1 and C2 segments, respec- represented within vchain transcripts, and identification of tively (4, 12). Comparison of the two deduced amino acid the vchain protein. The information presented here should sequences reveals four differences, no one ofwhich coincides be useful to these ends. with any one of the nine amino acid differences between the Note Added in Proof. Lefranc et al. (19) have recently presented the two sequenced murine CI exons (1). There is no obvious DNA sequence of several rearranged human T-cell v-chain V-J contribution of the CI exon to any functional difference regions found on genomic clones, two of which are productive between the two y-chain C-region gene segments. (in-frame) rearrangements. One of the V regions and one of the J From Fig. 3, it is apparent that amino acid homology regions presented by Lefranc et al. are identical at the amino acid between human and murine y-chain cDNA sequences ex- level to the V and J regions found in pTv1/2, as well as to an tends into the J region. The J region utilized by pTr1/2, unrearranged V region and an unrearranged J region found on however, is truncated by two amino acids at its 3' end relative genomic clones by Quertermous et al. (T.Q., W. Strauss, C. M., to that of pHDS4. The homology between pTy-1/2 and D.P.D., J.L.S. & J.G.S., unpublished observations). pHDS4 J regions for the most part extends to the J regions D.P.D. is pleased to acknowledge the valuable advice of Drs. Paul found in either murine or human T-cell receptor a- or p-chain Wong, Sam Speck, and particularly Doug Melton during construc- cDNAs (Fig. 5). Given that to date all murine -chain tion of cDNA library MLT:XgtlOA. D.P.D. was supported by a genomic and cDNA J-region sequences have been found to Damon Runyon-Walter Winchell postdoctoral fellowship. J.M.B. be identical at the amino acid level (1, 2), one a priori might was supported by National Institutes of Health Postdoctoral Fel- have expected the homology between pTr1/2 and pHDS4 J lowship F32 A106860. J.G.S. was supported by National Institutes of regions to be greater than that found (35%). Health Grants AI-19938 and AI-18436 and by a grant from the As only one of the three closely related murine -chain Mallinkrodt Foundation. J.L.S. was supported by National Institutes genomic V regions so far identified appears to be transcribed of Health Grant AM-30241. (1, 2), the murine -chain V region can thus far be opera- 1. Hayday, A. C., Saito, H., Gillies, S. D., Kranz, D. M., tionally defined as a constant segment. One might therefore Tanigawa, G., Eisen, H. N. & Tonegawa, S. (1985) Cell 40, have expected the homology between human and murine 259-269. y-chain V regions to be comparable to that found for the 2. Kranz, D. M., Saito, H., Heller, M., Takagaki, Y., Eisen, segments corresponding to the murine CI and CIII exons. H. N. & Tonegawa, S. (1985) Nature (London) 313, 752-755. The failure to identify a human y-chain genomic V region, 3. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) : A Laboratory Manual (Cold Spring Harbor Labora- using a murine --chain cDNA as probe, under conditions that tory, Cold Spring Harbor, NY). allowed identification of a human y-chain CI exon (4) sug- 4. Murre, C., Waldmann, R. A., Morton, C. C., Bongiovani, gested that this was not the case. That this is in fact not the K. F., Waldmann, T. A., Shows, T. B. & Seidman, J. G. case is indicated in Fig. 3, where it is apparent that the amino (1985) Nature (London) 316, 549-552. acid homology between human and murine y-chain V regions 5. Auffray, C. & Rougeon, F. (1980) Eur. J. Biochem. 107, is relatively poor. This is underscored by the observation that 303-314. the homology of the pT-1/2 V region with the murine 6. Hunyh, R. V., Young, R. A. & Davis, R. W. (1984) in DNA -chain V region (25%) is not much greater than that with the Cloning Techniques: A Practical Approach, ed. Glover, D. H. V region of the human T-cell receptor a (18%) or f (20%M) (IRL, Oxford). a 7. Vieira, J. & Messing, J. (1982) Gene 19, 259-268. chains and is in fact comparable to that with V region from 8. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, a murine T-cell receptor a-chain cDNA (24%) (Fig. 6). The 499-560. basis for the poor homology between human and murine 9. Biggin, M. D., Gibson, T. J. & Hong, G. F. (1983) Proc. Nati. ychain V regions is unclear, although the possibilities in- Acad. Sci. USA 80, 3963-3%5. clude fixation of different V regions that recognize either 10. Sanger, R., Nicklen, S. & Coulson, A. R. (1977) Proc. NatI. different epitopes on a highly conserved ligand or different Acad. Sci. USA 74, 5463-5467. epitopes on a highly divergent ligand. 11. Saito, H., Kranz, D. M., Takagaki, Y., Hayday, A. C., Eisen, Finally, a striking feature of the pTy-1/2 sequence pre- H. N. & Tonegawa, S. (1984) Nature (London) 309, 757-762. sented in Fig. 3 is the presence of five potential sites for 12. Quertermous, T., Murre, C., Dialynas, D., Duby, A., Strominger, J., Waldmann, T. & Seidman, J. G. (1985) Science N-linked glycosylation (N-X-S or N-X-T), four of which 231, 252-255. reside in the C region. This contrasts with the notable 13. Chien, Y., Becker, D. M., Lindsten, T., Okamura, M., Cohen, absence of such sites in the murine -chain sequence (11). D. I. & Davis, M. M. (1984) Nature (London) 312, 31-35. Their absence in the murine sequence argues against an 14. Chien, Y., Gascoigne, N. R. J., Kavaler, J., Lee, N. E. & absolute requirement for N-linked glycosylation in -chain Davis, M. M. (1984) Nature (London) 309, 322-326. function. The human -chain polypeptide has a greater 15. Jones, N., Leiden, J., Dialynas, D., Fraser, J., Clabby, M., predicted molecular weight (Mr 40,000) than does its murine Kishimoto, T., Strominger, J., Andrews, D., Lane, W. & counterpart (Mr 33,000) (11) due, in part, to the elongated Woody, J. (1985) Science 227, 311-314. CII exon evident in the sequence. Utiliza- 16. Saito, H., Kranz, D.M., Takagaki, Y., Hayday, A. C., Eisen, putative pT-1/2 H. N. & Tonegawa, S. (1984) Nature (London) 312, 36-40. tion of the potential sites for N-linked glycosylation in the 17. Yanagi, Y., Chan, A., Chin, M., Minden, M. & Mak, T. W. pT-1/2 sequence would result in an even greater difference (1985) Proc. Natl. Acad. Sci. USA 82, 3430-3434. in apparent molecular weight between the human and murine 18. Yanagi, Y., Yoshika, Y., Leggett, K., Clark, S. P., Alexander, -chain . I. & Mak, T. W. (1984) Nature (London) 308, 145-159. An understanding of human T-cell vchain function will 19. Lefranc, M. P., Forster, A. & Rabbitts, T. H. (1986) Nature require an analysis of somatic rearrangement of the vchain (London) 319, 420-422. Downloaded by guest on October 1, 2021